Search

Genetic immunisation is a simple method for producing polyclonal antibodies in mice. By this method, we produced antibodies against bovine interleukin-4 (BoIL-4). After a final injection with a recombinant BoIL-4 protein, nine stable hybridoma cell lines were established which secreted monoclonal antibodies (MAbs) against this cytokine. Specific binding of each of the MAbs to recombinant BoIL-4 produced by Escherichia coli, baculovirus, and Trypanosoma brucei was demonstrated in an indirect ELISA and/or in Western blotting. These MAbs recognise the same antigenic region localised in the first 47 amino acids of the mature protein. None of them was able to neutralise the biological activity of the BoIL-4 under the conditions tested but one allowed the detection of BoIL-4 by flow cytometry.

The eighteen monoclonal antibodies (mAbs) to B cells and the fourteen mAbs to accessory cells submitted to the workshop were analysed by FACS on three established, bovine leukemia virus (BLV)-infected bovine cell lines. Several mAbs of previously defined specificity were run in parallel. This analysis allowed us to gain further insight on the precise phenotype of those peculiar cells and to cluster the submitted mAbs according to their staining patterns. The BLV-infected cell lines seemed to belong to the B cell type though some of them lack detectable surface immunoglobulins. Moreover, all lines express the CD5 T cell marker and several myeloid markers.

A monoclonal antibody termed B2 Val 7C7, was produced by the fusion of xenoimmune mouse spleen cells with Sp2/0.Ag 14 myeloma cells. This antibody is specific for a polymorphic lymphocyte antigen; it was detected on cells from 138 out of 177 cattle by both 125I-labelled protein A (solid-phase radioimmunoassay, SPRIA) and gold-labelled protein A (immunogold). Its binding was tested on various cell types (peripheral blood lymphocytes, monocytes, polymorphonuclear cells (PMN), thymocytes) from a variety of normal bovine donors. On the one hand, B2 Val 7C7 detects a determinant present on all IgG-bearing lymphocytes, on 20% of the non-IgG-bearing lymphocytes and on the majority of the monocytes. On the other hand, no binding occurs on any PMN or thymocytes. The detected membrane antigen was isolated by immunoprecipitation from an NP 40 extract of 3H-leucine-labelled cells. On SDS-PAGE, it appears to be composed of two sub-units: a 32 000-dalton and a 27 000-dalton chain. These results show that B2 Val 7C7 recognizes an alloantigenic specificity present on an Ia-like antigen.

In the course of generating monoclonal antibodies to bovine thymus-dependent differentiation antigens, we were able to characterize an antibody, termed 8C11, that detects an antigen shared by a majority of thymocytes and peripheral T cells (in blood and thymus-dependent area of spleen and lymph-nodes), but undetectable on normal B cells. However, this antibody was reactive with B cells from cows infected with bovine leukemia virus (BLV). These BLV-infected B cells were found to express simultaneously high concentrations of both surface IgM and 8C11-detected antigen. The antigen recognized by this antibody was shown to be a 67.5 kDa molecule. Because similar findings have been made on mouse myelomas and on human chronic leukemia cells, where this antigen was considered to be the equivalent of mouse Ly-1 antigen and human Leu-1 or CD5 antigen, the T cell antigen detected on BLV-infected cells could be the bovine counterpart of the CD5 antigen. By another way, it has been found that the CD5 T cell antigen is also present on a minor subpopulation of B cells in the spleen but not in the blood. We suggest that in the bovine a similar B cell subpopulation should be the BLV target and expand as a consequence of viral insertion, leading to the persistent lymphocytosis observed on BLV-infected animals.

The amino acid sequence of the N-terminal 12 residues of papain digested BoLA class I molecules has been determined. This sequence except for its 5th amino acid, appears largely similar to those reported in man, mouse and rat.